Compositions for eliciting an immune response against Mycobacterium avium subspecies paratuberculosis

ABSTRACT

The invention provides compositions and method for stimulating an immunological response against  Mycobacterium avium  subspecies  paratuberculosis  (MPT). The compositions comprise at least five recombinant immunogenic components. The immunogenic components can be MPT antigens or DNA polynucleotides encoding MPT antigens, or combinations thereof. MPT antigens used in the invention include MPT 85A, 85B, 85C, 35 kDa, super oxide dismutase (SOD), MptC, MptD and ESAT-6 protein. The method comprises administering the composition to an animal in an amount effective to stimulate an immunological response against MPT bacteria. The method is of benefit to any animal susceptible to MPT infection, but is particularly beneficial for ruminants.

This application claims priority to U.S. provisional patent applicationSer. No. 60/653,536, filed Feb. 16, 2005, the disclosure of which isincorporated herein in its entirety by reference.

FIELD OF THE INVENTION

The present invention relates generally to stimulation of immunologicalresponses, and more specifically to compositions and methods forstimulating prophylactic or and therapeutic immunological responsesagainst Mycobacterium avium subspecies paratuberculosis.

BACKGROUND OF THE INVENTION

Mycobacterium avium subspecies paratuberculosis (MPT) is the causativeagent of Johne's disease (JD), which causes chronic granulomatousenteritis in ruminants. Clinically affected animals develop chronicdiarrhea and progressive weight loss that eventually results in death,while subclinically infected animals mainly have decreased production ofmilk. JD is of tremendous economic importance to the worldwide dairyindustry, causing major losses due to reduced production and earlyculling of animals with estimates of 20% of U.S. dairy herds affectedand costs of $220 million per year to the dairy industry (Wells, et al.2000. J. Am. Vet. Med. Assoc. 216:1450-1457). Cattle are mostsusceptible to infection with this organism within the first 6 months oflife, but disease typically does not become evident until 3 to 5 yearsof age. Infection occurs by ingestion of contaminated manure, colostrum,or milk from infected cows (Sweeney, 1996. Vet. Clin. N. Am. Food Anim.Pract. 12:305-312). Fetal infection also occurs, particularly inpregnant cows with advanced disease (Sweeney, et al. 1992. Am. J. Vet.Res. 53:477-80). Although JD is an important infectious disease ofruminants, there is no effective vaccine against this disease. The onlycurrently available vaccine in the United States consists of killed M.avium subsp. paratuberculosis in an oil adjuvant (Kormendy, B. 1992.Acta Vet. Hung. 40:171-184; Larsen, et l., 1978. Am. J. Vet. Res.39:65-69). However, such vaccination programs have raised serious publichealth concerns. For example, at least one veterinarian was accidentallyinoculated in the hand during vaccination of animals (Patterson, et al.,(1988) J. Am. Vet. Med. Assoc. 192:1197-1199). Further, studies havedemonstrated that there is a strong reaction at the injection sitesafter vaccination with this killed bacteria (Kormendy, B. 1992. ActaVet. Hung. 40:171-184; Larsen, et l., 1978. Am. J. Vet. Res. 39:65-69).Another drawback of this vaccine is that the vaccinated animals becometuberculin skin test positive (Kormendy, B. 1992. Acta Vet. Hung.40:171-184; Larsen, et l., 1978. Am. J. Vet. Res. 39:65-69). Thus, thereis a need for the development of more effective vaccines against JD thatcan be used as safe and effective prophylactic and/or therapeuticcompositions for MPT infection.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for stimulatingan immunological response in animals against MPT. The compositionscomprise immunogenic components which can be MPT antigens orpolynucleotides encoding for MPT antigens, or combinations thereof. Inone embodiment, the compositions comprise at least five recombinantimmunogenic components. Exemplary MPT antigens include MPT 85A, 85B,85C, 35 kDa, super oxide dismutase (SOD), MptC, MptD and ESAT-6 likeprotein.

The method comprises administering the composition to an animal in anamount effective to stimulate an immunological response against MPTbacteria. The method is of benefit to any animal susceptible to MPTinfection, but is particularly beneficial for ruminants.

Compositions comprising recombinant MPT protein antigens, DNApolynucleotides encoding MPT antigens, or combinations thereof can beformulated with standard pharmaceutical carriers and can be administeredvia any of a variety of conventional routes. The compositions can beadministered at any time to an animal susceptible to contracting MPTinfection or an animal that is infected with MPT. However, it ispreferable to administer the compositions of the invention prior to MPTinfection, such as by administration to pregnant animals who cantransfer prophylactic immunologic components to their newborns viacolostrum, or by administration during the period from one to five weeksafter birth.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of data from analysis ofproliferative responses of peripheral blood mononuclear cells frominfected and healthy control cows stimulated in vitro with 5 MPTrecombinant proteins. The results are expressed as a stimulation indexand the error bars represent standard deviation from the mean. Nosignificant proliferation was noted to any antigen by PBMCS fromnon-infected cows (P>0.05). 85A and the 35-kDa protein showed mostproliferative activity in low and medium shedders, respectively.

FIG. 2. is a graphical representation of data from analysis ofinterferon-γ production in response to individual antigens in related toMPT shedding levels. The results are given as O.D. values in stimulatedwells—O.D. values in control (naturally produced IFN-γ) wells. Errorbars represent standard deviations from the means. 85A and 85B were mostinducible antigens to produce IFN-γ in bovine peripheral bloodmononuclear cells from both shedders.

FIG. 3. is a graphical representation of data from analysis of antibodyresponses to individual antigens in relation to MPT shedding levels.Bars represent the means O.D. values at 405 nm. Error bars representstandard deviations from the means. All recombinant antigens showedincreases of antibody responses according to shedding levels andantibody responses to the 35-kDa protein were positively separatedbetween non-infected healthy cows and both shedders (P<0.01).

FIGS. 4A-4D are graphical representations of data from analysis ofchanges in T cell subset distribution in bovine peripheral bloodlymphocytes after stimulation with recombinant antigens, as determinedby FACS analysis. FIG. 4A. CD4; Ag 85A and Ag 85B induced a higherproportion of CD4⁺ T lymphocytes in medium shedders compared to lowshedders while the percentage of CD4⁺ lymphocytes was unchanged innon-infected control cattle. FIG. 4B. CD8; Ag 85A increased theproportion of CD8⁺ T lymphocytes in medium shedders, while the increasedpercentage of CD8⁺ lymphocytes was very low in non-infected cattle. FIG.4C. CD25; Ag 85A and Ag85B increased the proportion of CD25⁺ T cells inboth shedder groups while they had little effect in non-infected cattle.In contrast, Ag 85C and the 35-kDa protein significantly increased theproportion of CD25⁺ T cells only in the medium shedders (P<0.05). FIG.4D. γδ⁺ T-cells; all antigens resulted in significantly lower increasesin all cell subsets in both the low and medium shedder groups except SODfor γδ⁺ T cells in medium shedders.

FIG. 5 is a graphical representation of data from analysis ofdifferential changes of CD3⁺ T lymphocytes in response to stimulationwith recombinant proteins and two controls (ConA and PPD). Data areexpressed as the average of cells staining positive for CD3 (1 standarderror of the mean) in response to each recombinant antigen relative tothe shedding level.

FIG. 6 is a graphical representation of data from analysis of increasedCD21⁺ B lymphocyte subsets in bovine peripheral blood lymphocytes afterstimulation with recombinant antigens, as determined by FACS analysis.The results are reported as the average percent increase inpositive-staining cells and the error bars represent 1 standard error ofthe mean (SEM). Recombinant 35-kDa protein induced the largest increasein CD21⁺ B lymphocytes in medium shedders. No significant increase inthe proportion of B lymphocytes was observed in response to the otherantigens regardless of bacterial shedding levels (P>0.05).

FIG. 7. is a graphical representation of data from analysis of IL-2profiles of bovine PBMCs from non-infected cattle, low and mediumshedders after stimulation with recombinant antigens for 24 hrs. Resultsrepresent the mean fold increases of IL-4 over un-stimulated PBMCs,which served as calibrators. Ags 85 A and B most strongly stimulatedmedium shedders while the 35 kDa protein and SOD had lesser effects(p<0.05).

FIGS. 8A-8C are graphical representations of data from analysis ofcomparison of cytokine mRNA profiles for IFN-γ (FIG. 8A), IL-12p40 (FIG.8B) and TNF-α (FIG. 8C) of bovine PBMCs from non-infected cattle, lowand medium shedders after stimulation with recombinant antigens for 24hrs. Results represent the mean fold increase over un-stimulated PBMCs,which served as calibrators. The results are similar with Ags 85 A and Bmost strongly stimulating medium shedders while the 35 kDa protein andSOD had lesser effects.

FIG. 9. is a graphical representation of data from analysis of IL-4 mRNAprofiles of bovine PBMCs from non-infected cattle, low and mediumshedders after stimulation with recombinant antigens for 24 hrs. Resultsrepresent the mean fold increases of IL-4 over un-stimulated PBMCs,which served as calibrators. The 35-kDa protein strongly stimulated IL-4mRNA expression in both low and medium shedders.

FIG. 10 is a graphical representation of bacterial recovery from spleenand liver after administration of the indicated DNA constructs andcontrols and subsequent challenge with MPT.

FIG. 11 is a graphical representation of the mean number of granulomasin spleen and liver after administration of the indicated DNA constructsand controls and subsequent challenge with MPT.

FIGS. 12A and 12B are photographic representations of Ziehl-Neelsenstaining of tissues revealing numerous acid-fast bacilli (FIG. 12A). Incontrast, the infection was much less severe in the mice vaccinated withthe MPT DNA constructs encoding all five antigens (FIG. 12B).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for stimulatingan immunological response against MPT in an animal. The compositionscomprise DNA polynucleotides encoding MPT antigens, MPT antigens, orcombinations thereof. In one embodiment, at least five immunogeniccomponents are selected from recombinant MPT antigens and DNApolynucleotides. The method comprises administering the composition toan animal in an amount effective to stimulate an immunological responseagainst MPT bacteria.

As used herein, an “immunological component” is a component of thecomposition that can directly or indirectly stimulate an immunologicalresponse. Accordingly, when introduced into an animal, expressionvectors comprising DNA polynucleotides encoding MPT antigens enter cellsof the animal and express MPT antigens. The expressed MPT antigens inturn stimulate an immunological response. Thus, a DNA polynucleotideencoding an MPT antigen is considered an immunogenic component whichindirectly stimulates an immunological response. In respect of anadministered MPT protein antigen, since the antigen is recognizeddirectly by the immune system, the antigen is considered an immunogeniccomponent which directly stimulates an immunological response.

The method can provide benefit to any animal susceptible to MPTinfection, where infection is considered to mean colonization of theintestinal mucosa of the animal by MPT. However, the compositions andmethod are particularly well suited for prophylaxis or therapy for MPTinfection of ruminants, including but not limited to cattle, sheep,goats, deer and elk, antelope, and buffalo.

Thus, the compositions can be administered to any MPT infected ornon-infected animal. Administration of the compositions to infectedanimals according to the method of the invention is considered tostimulate a therapeutic immunological response. However, it ispreferable to administer the compositions prior to MPT infection tostimulate a prophylactic response. For example, the compositions can beadministration to a pregnant animal who can transfer prophylacticimmunologic components to their non-infected newborns via colostrum.Alternatively, the compositions can be administered during the periodfrom one to five weeks after birth to provide a prophylactic effectwhich can prevent infection or reduce the severity of disease ifinfection occurs. Thus, in one embodiment, the method of the inventionis prophylactic for MPT infection, while in another embodiment, themethod is therapeutic for MPT infection. The method can also be used forprophylaxis or therapy of Johne's Disease.

Suitable MPT antigens for use in the invention include but are notlimited to MPT proteins 85A, 85B, 85C, 35 kDa, SOD, MptC, MptD andESAT-6 like protein. Recombinant MPT protein antigens can be obtainedfor use in the invention by techniques known to those skilled in theart, such as by conventional recombinant cloning methods. Suitable DNAcloning procedures and methods for expressing and purifying recombinantproteins are known. (See, for example, Sambrook et al. 2001, Molecularcloning: a laboratory manual, 3rd ed. Cold Spring Harbor LaboratoryPress, New York, N.Y.). In general, to obtain recombinant MPT proteinantigens, MPT genomic DNA can be obtained from an MPT culture accordingto standard methods, such as by the well known alkaline lysis procedure.The DNA encoding the antigens can be amplified, such as by thepolymerase chain reaction, from the genomic DNA and the amplificationproducts can be cloned individually or in various combinations into aone or more suitable expression vectors. Appropriate host cells can betransfected with the expression vector and the transfected cells can becultured under appropriate conditions for expression of the antigens.The antigens can be subsequently extracted and purified from the cultureaccording to standard techniques.

For administration to animals, suitably purified recombinant MPTantigens can be combined with standard pharmaceutical carriers.Acceptable pharmaceutical carriers for use with proteins are describedin Remington's Pharmaceutical Sciences (18th Edition, A. R. Gennaro etal. Eds., Mack Publishing Co., Easton, Pa., 1990). Further, the antigensmay be provided in conventional liposomal or microsomal formulations.

Compositions comprising the MPT antigens for use in stimulating animmunological response can be administered by any acceptable route.Suitable routes of administration include oral, mucosal and parenteral(e.g., intravascular, intramuscular, and subcutaneous injection). Thoseskilled in the art will recognize that the amount of antigensadministered to a particular animal will depend on a number of factorssuch as the route of administration, and the size, physical conditionand the MPT status of the animal. The relative amounts of each antigenin a formulation can be adjusted according to known parameters, such asto provide molar equivalents or other ratios of the antigens. Further,the compositions can be used in a single administration or in a seriesof administrations to boost the immunological response to the MPTantigens. In general, a total dosage of between 10-200 μg of protein canbe administered. When DNA encoding MPT antigens is administered, ingeneral, between 30-500 μg of DNA can be administered.

In one embodiment, the method of the invention comprises administrationof a composition comprising at least five recombinant MPT antigens. Forexample, MPT antigens 85A, 85B, 85C, 35 kDa antigen and superoxidedismutase (SOD) can be administered in a single formulation. 85A, 85Band 85C are fibronectin-binding proteins; the 35 Kda and SOD proteinsare outer surface proteins. The DNA sequence encoding the MTP 85A geneand the amino acid sequence of the 85A gene, is provided in GenBankaccession no. AF280067 (Oct. 10, 2003, entry). The DNA sequence encodingthe MTP 85B gene and the amino acid sequence of the 85A gene is providedin GenBank accession no. AF219121 85B gene (Nov. 21, 2002 entry). TheDNA sequence encoding the MTP 85C gene and the amino acid sequence ofthe 85C gene is provided in GenBank accession no. AF280068 (Nov. 21,2002 entry). The DNA sequence encoding the MTP SOD gene and the aminoacid sequence of the SOD gene is provided in GenBank accession no.AF180816 (Nov. 30, 2001 entry). The DNA sequence encoding the 35 kDaprotein is provided herein as SEQ ID NO:1. The amino acid sequence ofthe 35 kDa protein is provided herein as SEQ ID NO:2. Additional MPTinclude MptC, MptD and ESAT-6 like protein. The DNA sequence encodingthe MptC protein is provided herein as SEQ ID NO:3. The amino acidsequence of the MptC protein is provided herein as SEQ ID NO:4. The DNAsequence encoding the ESAT-6 like protein is provided herein as SEQ IDNO:5. The amino acid sequence of the ESAT-6 like protein is providedherein as SEQ ID NO:6. The DNA sequence encoding the MptD sequence isprovided herein as SEQ ID NO:7. The DNA sequence encoding the MptD aminoacid sequence is provided herein as SEQ ID NO:8.

The DNA sequences of primers used to amplify DNA encoding the MPTantigens used herein from genomic MPT DNA are provided in Table 1.

TABLE 1 Accession No. of Length ss amplification SEQ of DNA product andamino Gene/ ID: Primer Sequence product acid sequence of primer name NO(5′->3′) (bp) antigen 85A pVR85AF  9 CGGGATCCATGATGACGCTTGTCGACA 1050AF280067 pVR85AR 10 CGGGATCCTTAGGTGCCGTGG 85B pVR85BF 11CGGGATCCATGACAGATCTG 1000 AF219121 pVR85BR 12 CGGGATCCTTATCCGCCGCC 85CpVR85CF 13 CGGGATCCATGTCGTTCATCGAA 1100 AF280068 pVR85CR 14CGGGATCCTCAGGTGGCGGGC SOD pVRSODF 15 GGATCCTGGGACTATGCAGC  590 AF180816pVRSODR 16 AGATCTTCAGCCGAAGATCAGGC 35 Kda (MAP2121c) pVR35KDF 17GGATGCCGACTTGGTGATGT  910 pVR35KDR 18 AGATCTTCACTTGTACTCATGGAACT MptC(MAP 3734) pVRMPTCF 19 GGATCCGGCGGTCGGCGT 1750 pVRMPTCR 20AGATCTTCATGGTCGAGGTGCCT MptD (MAP 3733) pVRMPTDF 21 GGATCCCGCCGCATCGAC 600 pVRMPTDR 22 AGATCTTCAAGCTAGGCGGGC ESAT 6 Like(MAP 0161) pVRESATF 23GGATCCCCGGGCGCGGTG  270 pVRESATR 24 AGATCTTCAGAACAGGCCG

In another embodiment, compositions comprising DNA polynucleotides whichencode five or more MPT antigens can be prepared. MPT antigen encodingsequences can be obtained by amplification of MPT genomic DNA usingappropriate primers and inserting the amplification products intoexpression vectors in the same manner as described for preparation ofrecombinant antigen proteins.

Suitable expression vectors contain appropriate eukaryotic transcriptionand translation signals, and may contain additional elements, such aspolyadenylation and/or protein trafficking signals. One example of asuitable expression vector is pVR1020 (available from Vical, Inc., SanDiego, Calif.), which contains an immediate-early cytomegaloviruspromoter to promote efficient expression in a eukaryotic host, as wellas a plasminogen activator secretion signal to facilitate secretion ofthe antigens from the cells of the eukaryotic host.

It will be recognized by those skilled in the art that one or moredistinct expression vectors, as distinguished from one another by theMPT antigens they encode and/or by their regulatory or other elements,such as polycloning sites, will be used in the instant method. Thus, asingle expression vector encoding at least five MPT antigens, or atleast five expression vectors each encoding a different MPT antigen, orcombinations of expression vectors each encoding at least one MPTantigen, can be used in the present method to deliver polynucleotidesencoding at least five MPT antigens.

In one embodiment, DNA polynucleotide sequences encoding MPT antigens85A, 85B, 85C, SOD, MptC, MptD, 35 kDa, and ESAT6-like proteins can beprovided in separate expression vectors that can be used for proteinexpression and purification and for administration in variouscombinations to animals for stimulating an immune response.

The expression vectors encoding the MPT antigens may be formulated inany pharmaceutically effective preparation for administration to theanimals. Such formulations may be, for example, a saline solution suchas phosphate buffered saline (PBS). It is preferred to utilizepharmaceutically acceptable formulations which also provide long-termstability of the DNA. Thus, it is preferable to remove and/or chelationtrace metal ions from the formulation buffers or from vials and closuresin which the DNA is stored to stabilize and protect the DNA duringstorage. In addition, inclusion of non-reducing free radical scavengers,such as ethanol or glycerol, is useful to prevent damage of the DNA fromfree radical production that may still occur, even in apparentlydemetalated solutions. Further, the DNA may be provided in conventionalliposomal or microsomal formulations.

There is no limitation to the route that the DNA polynucleotides of theinvention can be delivered, so long as their delivery stimulates animmunological response against MPT in the recipient animals.Accordingly, the DNA polynucleotides of the present invention can beadministered to the animal by any means known in the art, such asenteral and parenteral routes. These routes of delivery include but arenot limited to intramusclar injection, intraperitoneal injection,intravenous injection, and oral delivery. A preferred route isintramuscular.

In another embodiment, the composition of the invention comprises atleast five immunogenic components which are provided as a combination ofMPT protein antigens and DNA polynucleotides encoding MPT antigens. Suchcompositions can be obtained by combining the recombinant MPT antigensdescribed herein and the polynucleotides encoding MPT proteins describedherein. In this regard, the polynucleotide sequences can be present inone or more expression vectors and the MPT antigens can be provided asrecombinant proteins. Compositions comprising recombinant MPT antigensand polynucleotides encoding MPT antigens can be combined withconventional pharmaceutical carriers and administered as describedherein and/or according to standard techniques. Conventional liposomalor microsomal preparations of the recombinant MPT antigens andpolynucleotides encoding MPT antigens can be provided. Further, and aswill be recognized by those skilled in the art, whether or not thecompositions comprise recombinant antigens alone as the immunogeniccomponents, DNA polynucleotides alone as the immunogenic components, orcombinations of recombinant antigens and DNA polynucleotides encodingthe recombinant antigens as the immunogenic components, the compositionsof the invention may further comprise a suitable adjuvant.

Thus, and without intending to be bound by any particular theory,administration of the recombinant MPT antigens, polynucleotides encodingrecombinant antigens, and/or combinations thereof according to themethod of the invention is believed to stimulate an immunologicalresponse that can be prophylactic or therapeutic with respect to MPTinfection.

The following examples describe the various embodiments of thisinvention. These examples are illustrative and are not intended to berestrictive.

Example 1

This Example provides a comparison of distinct lymphoproliferationeffectis in response to stimulation with individual antigens.

To examine lymphoproliferative responses, five MPT recombinant antigens,85A, 85B, 85C, 35 kDa antigen and superoxide dismutase (SOD), wereanalyzed for their ability to elicit proliferative responses in PBMCsobtained from cows with different MPT shedding levels. For this andother Examples as indicated herein, a total of 38 Holstein cows, 2 to 3years old, were divided into 3 groups. Healthy controls (n=18) werenegative for MPT infection as determined by negative fecal culture andnegative IS900 PCR testing. The healthy controls came from a farm thathas been fecal culture and IS900 PCR negative for the past ten years.Positive animals were subdivided into low shedders (n=16) and mediumshedders based on the number of colony forming units (CFU)/gram of feces(n=4). Low shedders are considered animals with 1-30 CFU/gram of feces.Medium shedders are considered animals with between 31-300 CFU/gram offeces. Heavy shedders (>300 CFU/gm feces) were unavailable since theyare culled immediately from farms once they are identified. Fecalcultures and IS900 PCR testing to determine MPT infection status werepreformed as previously described (Shin et al, (2004) J. Vet. Diagn.Invest. 16:116-120).

For use in lymphprolifations assays, recombinant antigens 85A, 85B, 85C,35 kDa antigen and SOD were cloned and expressed using standardtechniques and as previously described. (Dheenadhayalan et al., (2002)DNA Seq. 13:287-294; Shin et al., (2004) J. Vet. Sci. 5:111-117) andpurified as previously described (Skeiky et al., (1998) J. Immunol.161:6171-6179). The antigens used in these Examples had negligible (10pg/ml) endotoxin in a Limulus amebocyte assay.

For isolation and culture of bovine peripheral blood mononuclear cells,peripheral blood (20 ml) of all cows was collected from the tail veinwith heparinized vacuum tubes. Isolation of lymphocytes from heparinizedblood was performed by differential centrifugation using HISTOPAQUE1.077 (Sigma). Twenty ml of heparinized whole blood was layered over 15ml HISTOPAQUE in a 50-ml sterile polypropylene tube (Falcon) and thencentrifuged at 1000×g for 30 min at room temperature. The plasma layerwas discarded and the mononuclear cell layer was carefully collected andwashed three times with phosphate-buffered saline (PBS, pH 7.2).Contaminating red blood cells were lysed with 0.87% ammonium KCl bufferby inverting for 2 min at room temperature, then immediately adding 30ml PBS.

The washed cell pellets were suspended in PBS and counted using ahemacytometer and trypan blue to determine percent viability.Differential cell counts consistently showed greater than 96%lymphocytes, 1% monocytes and less than 3% granulocytes in the cellsuspension.

The lymphocytes were resuspended at 2×10⁶/ml in RPMI 1640 containing 10%endotoxin free FCS (Cellect Gold; ICN Biomedicals, Inc., Costa Mesa,Calif.), 2 mM L-glutamine, 10 mM HEPES, 100 IU/ml Penicillin, 100 μg/mLstreptomycin and 50 μg/mL gentamycin (Sigma) and 250 ul were added toeither 96-well round-bottomed plates or flat-bottomed plates, dependingon the purposes of the experiment.

To investigate lymphocyte proliferation in response to the individualantigens, a blastogenesis assay was performed. Briefly, PBMCs wereinitially incubated in a 96-well flat-bottomed microplate for 3 days at37 C in a humidified atmosphere with 5% CO₂. Cultures were thenstimulated with Con A (10 μg/mL), purified protein derivative (PPD)(each positive controls) (10 μg/mL) or each purified recombinant protein(10 μg/mL) and 40 μl (1.0 μCi) of methyl-3H-thymidine (PerkinElmer LifeScience Inc, MA, USA) in culture medium were added to each well. Thecells were incubated for an additional 18 h in the same conditions, andthe cells were then harvested using a semi automatic cell harvester(Skatronas Liter Norway). Blastogenic activity was recorded as countsper minute (cpm) of radioactivity based on liquid scintillationcounting. Results were expressed as stimulation indices (SI) calculatedas follows:

${S\; I\mspace{14mu}\left( {{stimulation}\mspace{14mu}{Index}} \right)} = \frac{\begin{matrix}{\left( {C\; P\; M\mspace{14mu}{of}\mspace{14mu}{antigen}\mspace{14mu}{stimulated}\mspace{14mu}{positive}\mspace{14mu}{culture}} \right) -} \\\left( {C\; P\; M\mspace{14mu}{of}\mspace{14mu}{background}} \right)\end{matrix}}{\begin{matrix}{\left( {C\; P\; M\mspace{14mu}{of}\mspace{14mu}{control}\mspace{14mu}{culture}\mspace{14mu}{or}\mspace{14mu}{Negative}} \right) -} \\\left( {C\; P\; M\mspace{14mu}{of}\mspace{14mu}{background}} \right)\end{matrix}}$

For this and other Examples herein, statistical analysis of data wasperformed in Excel and GraPad Prism software package version 2.0.Differences between individual groups, antigens and cytokine geneexpression were analyzed with the Student's t-test. Differences wereconsidered significant if probability values of P<0.05 were obtained.

As demonstrated in FIG. 1, proliferative activities of bovine PBMCs frommedium shedder cows were higher than other groups in response to allrecombinant proteins and two positive controls (P<0.05) although therewas variation among individual cows. PBMCs from medium shedder cowstreated with 85A, 85B and the 35-kDa protein antigens demonstrated astimulation index (SI) significantly higher (P<0.005) than that of PBMCstreated with other recombinant antigens. In addition, proliferativeresponses to the 35 kDa protein in low shedders were even greater thanthose of medium shedders in response to 85C and SOD (FIG. 1). Thus, thisExample indicates that the 85A, 85B and the 35-kDa protein antigens maybe important in affecting lymphocyte proliferation is animals previouslyexposed to MPT.

Example 2

This Example demonstrates the effects of recombinant antigens on IFN-γproduction. To analyze IFN-γ production, IFN-γ levels were measured inculture supernatants using a commercial kit specific for bovine IFN-γfollowing the manufacturer's instructions (Biosource Int. Camarillo,Calif.). The plates were read at 450 nm in a Bio-Tek 312E ELISA reader(BioTEK Instruments, Inc, Winooski, Vt. 05404-0998), using any referencefilter from 630 nm to 750 nm. The results were calculated based oncomparison of negative and positive control optional density (O.D).Results were determined as either negative (<OD of positive control) orpositive (>OD of positive control) relative to the cut-off valueaccording to the manufacturer's instructions.

IFN-γ production after stimulation with the five recombinant antigens ortwo positive controls was measured in PBMCs from infected and uninfectedcontrol cows. The results are presented in FIG. 2 as corrected OD values(OD of antigen stimulated minus OD of control) representing theelevation of IFN-γ production by the various antigens.

As can be seen from FIG. 2, all recombinant antigens tested inducedsignificant release of IFN-γ in cultures of bovine PBMCs from infectedcattle compared to uninfected controls (P<0.05), and IFN-g levels wereconsistently higher in medium shedders than in low shedders (P<0.05).The recombinant antigens 85A and 85B induced significantly higher levelsof IFN-γ in the low shedders than the other recombinant antigens testedand as compared to the two positive controls (P<0.05). Thus, thisExample indicates the recombinant antigens 85A and 85B may be importantin stimulating a cell-mediated response against MPT.

Example 3

This Example provides a comparison of antibodies in sera isolated fromnon-infected and infected cows which recognize the recombinant antigensof the invention.

Enzyme-linked immunosorbent assays (ELISA) were performed to evaluatethe seroreactivity of the recombinant antigens following steps aspreviously described (Shin, et al., (2004) J. Vet. Sci. 5:111-117).Briefly, an indirect ELISA was optimized using 2.5, 5 or 10 μg/mL ofeach antigen and 1:100 diluted serum by checkerboard titration.Flat-bottomed 96-well plates (Maxisorp, Nunc, Denmark) were coated with100 μL of each antigen in carbonate-bicarbonate buffer (14.2 mM Na₂CO₃,34.9 mM NaHCO₃, 3.1 mM NaN₃, pH 9.5) at 4° C. overnight, followed bywashing three times with PBS containing 0.05% Tween 20 (PBST, washingbuffer) using a microwell plate washer Bio-Tek ELx405 (BioTEKInstruments, Inc, Winooski, Vt.). Uncoated sites in the wells wereblocked with 5% skim milk in PBST at 37° C. for 1 h. The plates werewashed twice with PBST and 100 μL of optimally diluted (1:25,000)conjugated anti-bovine IgG-HRP (Sigma) was added to all wells andincubated at 37° C. for 1 h. The plates were washed three times in PBSTand 200 μL of 2-2′-Azino-Bis-Thiazoline-6-Sulfonic acid (Sigma) wasadded to each well. The plates were incubated at 37° C. in the dark.After 30 min incubation, stop solution (1M HCl) was added and the plateswere read 3 times at 405 nm at 2-minute intervals in a Bio-Tek 312 ELISAreader (BioTEK Instruments, Inc, Winooski, Vt. 05404-0998). Positive andnegative sera and antigen and antibody controls were included in eachplate.

The results from measuring levels of IgG antibodies to the recombinantantigens in sera from both shedder groups and healthy controls aredepicted in FIG. 3. Although there was a wide variation in antibodycontent in sera from individual cows, the mean IgG antibody responsesagainst all recombinant antigens increased significantly in both the lowand medium shedder groups. No significant differences were observedamong the mean levels of antibody of the low shedder group to any of theantigens tested (FIG. 3). Strikingly, antibody responses to the 35-kDaprotein were significantly higher in the medium shedder group than thoseto the other antigens (P<0.05), which may be important because the35-kDa protein is also effective at stimulating lymphocyteproliferation, and thus may stimulate both cell-mediated and humoralimmune responses.

Example 4

This Example demonstrates changes in lymphocyte subset distribution inPBMCs obtained from non-infected, low shedder and medium shedder cows inresponse to stimulation with the recombinant antigens.

To perform this analysis, a single-color flow cytometric analysis wasperformed with monoclonal antibodies against bovine lymphocyte markers(Table 2). Briefly, cells were washed three times in FACS buffer,incubated with the first

TABLE 2 Monoclonal antibody Isotype Antigen identified Ab ReferenceIL-A11 IgG2a CD4 (Brodersen, et al., 1998. Vet. Immunol. Immunopathol.64: 1-13) CACT80C IgG1 CD8α (Davis, et al. 1989. Am. Fish. Soc. Symp. 7:521-540.) MM1A IgG1 CD3 (Rhodes, et al. 2001. J. Immunol. 166:5604-5610) BAQ15A IgM CD21 B cells (Mukwedeya, et al. 1993. Vet ImmunolImmunopathol 39: 177-186) CACT116A IgG1 CD25 (IL-2Ra) (Naessenset al.1992. Immunology 76: 305-309.) CACT63A IgG1 γδ T cells (Davis, et al.1996. Vet. Immunol. Immunopathol. 52: 301-311)antibody (Table 1) for 30 min at 4 C, washed three times, subsequentlyincubated with a fluorescein isothiocyanate-labeled horse anti-mouseimmunoglobulin antibody (Vector) for 30 min at 4 C, washed twice, andcollected in 200 μl of FACS fixer buffer prior to analysis. Analysis wasdone on a flow cytometer (FACSCalibur; Becton Dickson). Aforward-scatter-side-scatter live gate was used to measure 5,000 to10,000 lymphocytes per sample. Based on the florescence data of thelymphocytes, the results were expressed as the percentage of cells withpositive staining relative to a sample stained with an irrelevantisotype control antibody.

As depicted in FIGS. 4A-4D, antigen-stimulated T cell and/or B cellsubsets were examined by single color flow cytometry for differences inthe percentage of CD4⁺, CD8⁺, CD3⁺ (CD3⁺ is depicted in FIG. 5), CD21⁺and CD25⁺ lymphocyte subsets, as well as γδ⁺ T cells in PBMC culturesfrom both shedder groups and healthy controls after stimulation witheach recombinant antigen (FIGS. 4 and 5). All lymphocyte subsetsinvestigated in this study increased but, depending on bacterialshedding levels, there were slight differences (P<0.05) betweennon-infected cattle and low shedders according to recombinant antigens.

CD3 is a pan T-cell marker that is expressed by CD4⁺ and CD8⁺ cells aswell as γσ⁺ T cells. The proportion of CD25⁺ T cells increased inculture regardless of the recombinant antigen used (P<0.05) (FIG. 4C).These results suggest that all antigens used in this study are able tostimulate sensitized T cells.

While all recombinant antigens tested increased the proportion of CD4⁺cells in cultures of bovine PBMCs from infected cattle compared touninfected controls (P<0.05), 85A and 85B increased the proportion ofCD4⁺ T cells to significantly higher levels than 85C, the 35-kDaprotein, and SOD (P<0.05) (FIG. 4A). The proportion of CD4⁺ T cells wasalso greater in cultures treated with 85A and 85B among PBMCs frommedium shedders than from low shedders (P<0.05) (FIG. 4A). Further, 85Aand 85B did not increase CD4⁺ T cells in noninfected cattle. While notintending to be bound by any particular theory, these results mayindicate that 85A and 85B antigens induce CD4⁺ T cells specificallysensitized by MTP and provide protective immunity against MTP infectionby maintaining circulating CD4⁺ T-cell populations in the earlyinfectious phase, i.e., at the time mucosal colonization by MPT is firstoccurring.

In contrast to the increase of CD4⁺ cells induced by all the antigensrelative to uninfected controls, a significant increase in theproportion of CD8⁺ T cells was found only in cultures treated with 85A,85B, and 85C and the proportion of CD8⁺ cells was also greater incultures treated with 85A and 85B among PBMCs from medium shedders thanfrom low shedders (P<0.05) (FIG. 4B). Thus, these antigens maypreferentially stimulate cell mediated responses.

Only SOD was able to significantly increase the proportion of γσ⁺ Tcells in the cultures of medium shedders (P<0.05) (FIG. 4D). However,SOD stimulated lymphocytes to a lesser degree than the other antigenstested, except for γσ⁺ T lymphocytes, as the number of γσ⁺ T cells wassignificantly higher in PBMC cultures treated with SOD in noninfectedcattle, as well as in both shedder groups (FIG. 4D). Thus, SOD maypreferentially stimulate γσ⁺ T lymphocytes compared to the otherantigens. Further, because γσ⁺ T cells are numerous in mucosal tissues,which is the point of entry for mycobacterial pathogens, the SOD antigenmay be important in the earlier stages of infection via its preferentialstimulation of γσ⁺ T cells.

All recombinant antigens tested significantly increased the proportionof CD21⁺ B cells in cultures of bovine PBMCs from both low and mediumshedders compared to uninfected controls (P 0.05) (FIG. 6).Interestingly, the proportion of CD21⁺ B cells was significantly higherin cultures of bovine PBMCs from medium shedders than that of the otherrecombinant antigens tested (P<0.05).

Example 5

This Example provides a comparison of stimulation of cytokine mRNAproduction in bovine PBMC's after stimulation with recombinant antigens.

For preparation of RNA and DNase I treatment of cells, PBMC cell pelletswere obtained and washed twice in 50 mL phosphate buffered saline (PBS),pelleted and 5×10⁶ cells were lysed with 350 μL lysis buffer accordingto the manufacturer's recommendations (RNeasy mini kit, Qiagen, Calif.)and kept at −80 C until RNA extraction and complementary DNA (cDNA)synthesis. Total RNA (tRNA) was extracted from lysed cells or PMBCsusing the RNeasy mini kit (Qiagen). The extracted tRNA was treated with10 U/μl of RNase-free DNase I at 37 C for 10 min followed by heatinactivation at 95 C for 5 min and then chilled on ice.

For cDNA synthesis, reverse transcription (RT) was performed in a 20 μLfinal volume containing 1.6 μL total RNA, 200 U Superscript II RT(GibcoBRL), 50 mM Tris-Hcl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 0.01 M DTT,and 0.5 mM dNTPs. The reaction mix was subjected to 42 C for 50 min andinactivated at 70 C for 15 min. The cDNA was analyzed immediately orstored at −20 C until use.

To perform real-time quantitative (RT-PCR), approximately 1 to 5 μg oftotal RNA from each treatment group was reverse transcribed by usingSuperscript reverse transcriptase, Random Hexamers, and reversetranscriptase reagents (Gibco BRL). Real-time primers and probes weredesigned by Primer Express software (Applied Biosystems) using thesequences for bovine GAPDH, cytokines and growth factors obtained fromGenbank. The internal probes were labeled with the florescent reporterdye 5-carboxyfloroscein (FAM) on the 5′ end and the quencher dyeN′,N′,N′,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA) on the 3′ end. ThePCR mixture consisted of 400 nM primers, 80 nM Taqman probe andcommercially available PCR Mastermix (TaqMan Univeral PCR Mastermix,Applied Biosytems) containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mMMgCl₂, 2.5 mM deoxynucleotide triphosphates, 0.625 U AmpliTaq Gold DNApolymerase per reaction, 0.25 U AmpErasw UNG per reaction and 10 μl ofthe diluted cDNA sample in a final volume of 25 μl. The samples wereplaced in 96-well plates and amplified in an automated flurometer (ABIPrism 7700 Sequence Detection System, Applied Biosystems). Ampliconconditions were 2 min at 50 C, 10 min at 95 C, followed by 40 cycles at95 C for 15 s and 60 C for I min. Final quantitation was done using thecomparative cycle threshold (C_(T)) method and is reported as relativetranscription or the n-fold difference relative to a calibrator cDNA.

As can be seen from FIG. 7, all recombinant antigens stimulated highlevels of IL-2 mRNA from PBMCs of medium shedders, with the antigen 85complex having a greater effect than either the 35-kDa protein or SOD(P<0.05) (FIG. 7). 85A also induced a high level of IFN-γ, IL-12p40, andTNF-α mRNA (FIGS. 8A, B, and C, respectively) in medium shedders(P<0.05). Strikingly, PBMCs stimulated with the 35-kDa protein antigenhighly expressed IL-4 mRNA in both low and medium shedders (P<0.05)(FIG. 9). This induction of IL-4 mRNA by the 35-kDa proteinsignificantly increased depending on shedding levels (P<0.001). Incontrast, no significant differences were observed among the otherantigens (P>0.05). These studies are in agreement with the resultspresented in Example 3 which indicate immune responses to the 35-kDaprotein may be more important in the later stage of disease since flowcytometric analysis showed that the 35-kDa protein strongly inducedproliferation of B lymphocytes, especially in medium shedders (FIG. 3).Thus, this Example demonstrates that all of the recombinant antigenstested may stimulate a cell-mediated immunological response.

Example 6

This Example demonstrates the effect of vaccinating mice with DNApolynucleotides encoding MPT antigens and subsequent challenge with MPT.

For demonstrating of the effect of the DNA constructs,specific-pathogen-free C57/BL6 female mice were obtained from the HarlanSprague Dawley Inc (Indianapolis, Ind.). The mice were ˜8 weeks old atthe time of vaccination. There were five groups of mice and each vaccinegroup consists of 25 animals during this experiment. The animals werefed commercial mouse chow and water ad libitum, and maintained on a12/12-hour light/dark cycle.

The commercially available eukaryotic expression plasmid pVR1020 (Vical,Inc., San Diego, Calif.) was used for the DNA vaccine. This plasmidcontains an immediate-early cytomegalovirus promoter to ensure efficientexpression in a eukaryotic host as well as the human tissue plasminogenactivator (hTPA) secretion signal to facilitate secretion of the targetantigen from the eukaryotic cell (Brandt, et al. 2000. Infect. Immun.68:791-795). DNA encoding MPT 85A, 85B, 85C, SOD, 35 kDa, 35 kDa(li),MptC, MptD, and ESAT-6 like genes was amplified by polymerase chainreaction from MPT genomic DNA using the gene specific primers listed inTable 1. Briefly, the primers used for amplification of MPT 85A, 85B and85C coding sequences each included a BamHI site. One primer foramplification of SOD, MptC, MptD, 35 kDa, and ESAT6-like codingsequences included a BamHI site while the other included a BglII site.The amplification products were digested using the indicated restrictionenzymes and cloned into the commercially available pCR2.1 TOPO cloningvector (Invitrogen, CA) using standard techniques. The MPT genes werethen subcloned downstream of the human tissue plasminogen activator(hTPA) secretion signal in the pVR1020 plasmid (Vical, Inc., San Diego,Calif.) using standard techniques and essentially as previouslydescribed (Dheenadhayalan et al., (2002) DNA Seq. 13:287-294; Shin etal., (2004) J. Vet. Sci. 5:111-117) (Skeiky et al., (1998) J. Immunol.161:6171-6179). These recombinant constructs were transfected intoHEK-293 (human embryonic kidney) cells with using Lipofectamin™ 2000transfection reagent (Invitrogen, CA) and the expression of the antigengenes was confirmed at the transcription level using RT-PCR.

For immunization and MPT challenge, mice were divided into fivedifferent groups (Table 3). The animals were administered with 50 μg ofeach DNA in 50 μl

TABLE 3 Groups 1 2 3 4 5 DNA 85A, 85B Group1 + MptC, MptD Group3 +Vector Vaccine 85C, IL-12 ESAT6like IL-12 Control 35 kDa and (pVR1020)and SOD 35 kDa(Li)PBS per dose via intramuscular injection. Mice were immunized threetimes at 3-week intervals. IL-12 genes were additionally injected asindicated in Table 3. Three weeks after the second boosters, the animalswere challenged by intraperitoneal injection of 10⁹ CFU units of (MPT).Six animals in each group were sacrificed at 4^(th), 8^(th), 12^(th) and16^(th) week after challenge and the recovery of bacteria from organs(liver, spleen, mesenteric lymph node, lung) was enumerated on Herald'sEggYolk (HEY) slant agar supplemented with Mycobactin J and antibioticsas previously described (Kamath, et al. Infect. Immun. 67:1702-1707).After challenge, feces were also collected every week from mouse cagesand cultured using the same agar. Tissues including liver, spleen, lung,intestine and mesenteric lymph node were fixed by immersion in 10%buffered formalin and processed for histopathological examination usingstandard histotechnology techniques. The presence of MPT (acid-fastbacteria) in the liver and spleen of each mouse was assessed byZiehl-Neelsen staining.

FIG. 10 shows the decreased mycobacterial burden in the livers andspleens of vaccinated mice relative to controls at 4, 8 and 12 weekspost-challenge and demonstrates an approximately 90% reduction (1 log10) in the bacterial burden in the spleens and livers for micevaccinated with the MPT DNA vaccine cocktail compared to non-immunizedcontrols. The relative liver and spleen histopathology data at 4, 8 and12 weeks post-challenge paralleled the bacterial growth results.Substantive differences were seen in liver and spleen tissues taken fromanimals immunized with the plasmid cocktail compared to nonimmunizedmice (FIG. 11). MPT infected nonvaccinated controls had numerousrandomly dispersed granulomas with central epithelioid macrophagessurrounded by small lymphocytes. Ziehl-Neelsen staining revealednumerous acid-fast bacilli were seen (FIG. 12A and inset). In contrast,the infection was much less severe in the mice vaccinated with the MPTDNA vaccine cocktail (FIG. 12B).

Thus, this Example demonstrates that administration with a DNAexpression vectors encoding at least five MPT antigens can providesignificant protection from MPT infection.

1. A method of stimulating an immune response against MycobacteriumAvium Subspecies Paratuberculosis (MPT) in a ruminant comprisingadministering an amount of a cocktail composition comprising at leastfive immunogenic components, wherein the immunogenic components areisolated polynucleotide sequences encoding full length MPT proteinantigens 85A, 85B, 85C, MPT 35 kDa protein, and superoxide dismutase(SOD).
 2. The method of claim 1, wherein the ruminant is a bovine, asheep, a goat, a deer or an elk.
 3. The method of claim 2, wherein theruminant is a bovine.
 4. The method of claim 1, wherein the ruminant isnot infected with MPT.
 5. The method of claim 1, wherein the ruminant isinfected with MPT.
 6. The method of claim 2, wherein the bovine hasJohne's Disease.
 7. The method of claim 1, wherein the compositionfurther comprises a pharmaceutically acceptable carrier.
 8. The methodof claim 1, wherein the ruminant is pregnant.
 9. The method of claim 7,wherein the composition further comprises an adjuvant.